Molecular sites of regulation of expression of the rat cationic amino acid transporter gene.

Cat-1 is a protein with a dual function, a high affinity, low capacity cationic amino acid transporter of the y+ system and the receptor for the ecotropic retrovirus. We have suggested that Cat-1 is required in the regenerating liver for the transport of cationic amino acids and polyamines in the late G1 phase, a process that is essential for liver cells to enter mitosis. In our earlier studies we had shown that the cat-1 gene is silent in the quiescent liver but is induced in response to hormones, insulin, and glucocorticoids, and partial hepatectomy. Here we demonstrate that cat-1 is a classic delayed early growth response gene in the regenerating liver, since induction of its expression is sensitive to cycloheximide, indicating that protein synthesis is required. The peak of accumulation of the cat-1 mRNA (9-fold) by 3 h was not associated with increased transcriptional activity of the cat-1 gene in the regenerating liver, indicating post-transcriptional regulation of expression of this gene. Induction of the cat-1 gene results in the accumulation of two mRNA species (7.9 and 3.4 kilobase pairs (kb)). Both mRNAs hybridize with the previously described rat cat-1/2.9-kb cDNA clone. However, the 3' end of a longer rat cat-1 cDNA (rat cat-1/6.5-kb) hybridizes only to the 7.9-kb mRNA transcript. Sequence analysis of this clone indicated that the two mRNA species result from the use of alternative polyadenylation signals. The 6. 5-kb clone contains a number of AT-rich mRNA destabilizing sequences which is reflected in the half-life of the cat-1 mRNAs (90 min for 7. 9-kb mRNA and 250 min for 3.4-kb mRNA). Treatment of rats with cycloheximide superinduces the level of the 7.9-kb cat-1 mRNA in the kidney, spleen, and brain, but not in the liver, suggesting that cell type-specific labile factors are involved in its regulation. We conclude that the need for protein synthesis for induction of the cat-1 mRNA, the short lived nature of the mRNAs, and the multiple sites for regulation of gene expression indicate a tight control of expression of the cat-1 gene within the regenerating liver and suggest that y+ cationic amino acid transport in liver cells is regulated at the molecular level.